The invention relates to a new microparticle enhanced light scattering agglutination assay for determining the amount of an analyte, and a microparticle reagent for performing that assay.
Microparticle agglutination tests were first described for the detection of rheumatoid factors by Singer J. and Plotz C., 1956, Am. J. Med. 21, 888-892, following the advent of reliable production methods for producing uniform latex particles of a wide range of sizes. Detection of the agglutination reaction by turbidimetry or nephelometry then made possible the development of truly quantitative microparticle enhanced light scattering agglutination tests, as described by Dezelic G. et al., 1971, Eur. J. Biochem. 20, 553-560, and Grange J. et al., 1977, J. Immunol. Methods, 18, 365-75.
The microparticle enhanced light scattering agglutination tests are quasi-homogeneous and do not need a separation and washing step at all. They thus meet the requirements for the automation with commonly used clinical chemistry analyzers or dedicated nephelometers. Such tests provide an increased sensitivity by a factor of 2 to 3 orders of magnitude (down to 10.sup.-11 mol/l analyte) compared to direct agglutination tests not using microparticles and, in addition, less matrix interference and higher flexibility.
Due to the favorable characteristics described above, the microparticle enhanced light scattering agglutination tests are now routinely used for quantifying proteins such as tumor markers (see for example Eda S. et al., 1993, Japanese J. Clin. Chem. 22, 99-103, or, 1992, in "Progress in Clinical Biochemistry" K. Miyai et al., pp. 265-267, Elsevier Publishers, Amsterdam, The Netherlands), specific proteins (see for example Winkes J. W. et al., 1989, Clin. Chem. 35/2, 303-307 or Chirot L. et al., 1992, Ann. Biol. Chim. 50, 143-147), drugs of abuse (see for example Ambruster D. et al., 1992, J. Anal. Toxicol. 16, 172-175) and therapeutic drugs (see for example "RDS Method Manual COBAS.RTM. INTEGRAL.RTM. 1996: Digoxin", F. Hoffmann-La Roche A.G., Basle, Switzerland).
However a drawback of microparticle enhanced light scattering agglutination assays is their limited dynamic range. Dynamic range, defined as the ratio of the upper measuring limit to the detection limit, is usually for those assays of only two orders of magnitude. Due to this limited dynamic range, the initial measurement often fails, requiring re-testing, under different dilution degrees of samples. The limited dynamic range thus causes additional expenses and loss of time, both of which are critical in laboratories performing those assays.
The problem addressed by the invention is therefore to provide a microparticle enhanced light scattering agglutination assay, and a microparticle reagent for performing that assay, that offer a larger dynamic range than hitherto known microparticle enhanced light scattering agglutination tests.
U.S. Pat. No. 4,595,661 describes a heterogeneous sandwich immunoassay wherein the hook effect, that is, a decrease of the signal at high antigen concentrations; is avoided by using an insoluble catcher antibody,and two soluble tracer antibodies having different affinities and different specificities to the antigen, the antibody of a lesser affinity making a significant contribution only at high antigen concentrations and thus forestalling the hook effect. That document states that the two exemplified assays according to the invention have the same dynamic range as those of the prior art (see column 6, lines 42-43 and column 8, lines 14-15).
PCT Patent Publication No. 89/11101 relates to an assay by flow cytometry, which uses the two distinguishable particles, for example particles of different sizes, as solid phase carriers of immunological binding partners having the same specificity but a different affinity for the same analyte. Different sizes of carrier particles are discriminated after separation in the capillary of the flow cytometry analyzer due to their different light scattering characteristics, which allows generation of two standard curves. That document and a subsequent publication of the inventor, T. Lindmo, 1990, J. Immunol. Methods 126, 183-189, specifically describe an assay for carcinoembryonic antigen (CEA) which uses particles of 7 .mu.m or 10 .mu.m diameter, respectively coated with a high affinity antibody or a low affinity antibody which bind to the same epitope, and a soluble labeled third antibody as a conjugate directed against another epitope. The flow cytometer records the fluorescence intensity of the conjugate bound on both particle types, and plots two separate standard curves. The system allows for a high dynamic range using sophisticated instrumentation and meticulously designed powerful analytical software which enable analyzing the data as if two immunoassays were run independently in parallel, one with particles of 7 .mu.m diameter coated with a high affinity antibody, which preferably binds the antigen at first and whose standard curve works at low concentrations of analyte, and another with particles of 10 .mu.m diameter coated with a low affinity antibody, whose standard curve works after the first standard curve flattens off.
Assays by flow cytometry and microparticle enhanced light scattering agglutination assays are based on totally different principles. In assays by flow cytometry there is no aggregation of microparticles and the amount of soluble labeled antibody is determined for each particle individually as they are separated and possibly, if they have distinguishing features, for example due to different sizes, discriminated by the flow cytometer; as many calibration curves are generated as there are particles with distinguishing features. In the microparticle enhanced light scattering agglutination assays there is a measuring as a whole, for example by turbidimetry or nephelometry, of the aggregation of binding partners bound to microparticles and analyte, without any possibility of determining the individual contribution of each particle or discriminating between particles having distinguishing features, and as a result only one calibration curve is generated.
The present invention will be further illustrated by the following examples. The following description will be better understood by referring to the following FIGS. 1A1B, 1C, 1D, 2, 3 A, 3B and 4.